A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential

Abstract

Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD and possibly affects mitochondrial function. This study aimed to elucidate the link between mitochondrial dysregulation and idiopathic ASD by focusing on mitochondrial respiratory capacity and membrane potential. Our findings showed that mitochondrial function in the energy metabolism pathway was significantly dysregulated in a lymphoblastoid cell line (LCL) derived from an autistic child (ALCL). Respiratory capacities of oxidative phosphorylation (OXPHOS), electron transfer of the Complex I and Complex II linked pathways, membrane potential, and Complex IV activity of the ALCL were analyzed and compared with control cell lines derived from a developmentally normal non-autistic sibling (NALCL). All experiments were performed using high-resolution respirometry. Respiratory capacities of OXPHOS, electron transfer of the Complex I- and Complex II-linked pathways, and Complex IV activity of the ALCL were significantly higher compared to healthy controls. Mitochondrial membrane potential was also significantly higher, measured in the Complex II-linked pathway during LEAK respiration and OXPHOS. These results indicate the abnormalities in mitochondrial respiratory control linking mitochondrial function with autism. Correlating mitochondrial dysfunction and autism is important for a better understanding of ASD pathogenesis in order to produce effective interventions.

Keywords: autism; high-resolution respirometry; mitochondria; mitochondrial membrane potential; mitochondrial respiration; oxidative phosphorylation.

Figure 1

Cytochrome c oxidase (CIV) activities of NALCL and ALCL. (a,b) are the protocols for measuring CIV activities in NALCL and ALCL, respectively. Titrations: Cell, dig (digitonin, cell permeabilization), Ama (antimycin A, CIII inhibition), CCCP (oxidative phosphorylation uncoupling), As (ascorbate, maintaining TMPD in a reduced state), TMPD (reducing cytochrome c), c (cytochrome c integrity of outer mt-membrane), and Azd (azide, CIV inhibition). Blue plots indicate O₂ concentration and red plots are the O₂ consumption expressed per cell. (c) The total O₂ consumption rate was baseline-corrected for autoxidation after inhibition of CIV. Results are expressed as means ± S.D. * denotes p < 0.05 compared to NALCL as determined by t-test.

Figure 2

Mitochondrial respiration of NALCL and ALCL. (a,b) are the protocols for measuring respiration in NALCL and ALCL, respectively. Titrations: Cell, dig (digitonin, cell permeabilization), P and M (pyruvate and malate, non-phosphorylating N-LEAK respiration, N(GM)_L(n)), ADP (N-OXPHOS capacity, N(GM)_P), c (cytochrome c integrity of outer mt-membrane), G (glutamate, N-OXPHOS capacity, N(PGM)_P), S (succinate, NS-OXPHOS, NS_P), CCCP (NS-ET capacity, NS_E), Rot (rotenone, CI inhibition, S-ET capacity, S_E), and Ama (antimycin A, CIII inhibition, Rox). Oxygen consumption was corrected for Rox. Blue plots indicate O₂ concentration and red plots are the O₂ consumption expressed per cell. (c) The Rox-corrected respiration: ROUTINE respiration, R, was measured in non-permeabilized cells in MiR05. After plasma membrane permeabilization, five respiratory states were sequentially established to measure NADH-linked LEAK respiration with pyruvate and malate, N_L, OXPHOS capacity, N_P, NS-pathway OXPHOS capacity, NS_P, and NS- and S-pathway ET capacity, NS_E and S_E, where S indicates the succinate pathway. Results are expressed as means ± S.D. * denotes p < 0.05 while ** denotes p < 0.01 compared to NALCL as determined by t-test.

Figure 3

Succinate-linked respiration of NALCL and ALCL measured simultaneously with mitochondrial membrane potential in the presence of safranin. (a,b) are the protocols for measuring respiration in NALCL and ALCL, respectively, while (c,d) are the protocols for measuring mitochondrial membrane potential, mtMP using safranin concentration in NALCL and ALCL, respectively. Before the cells were added, safranin was calibrated up to 2 µM. The initial drop to 1.6 µM safranin is mainly due to unspecific binding upon the addition of cells. Low safranin concentrations indicate high mtMP. Titrations: Cell, dig (digitonin, cell permeabilization), Rot (rotenone, CI inhibition), S (succinate, S-LEAK respiration, S_L(n)), ADP (S-OXPHOS capacity, S_P), Omy (oligomycin, S-LEAK, S_L(Omy)), CCCP (S-ET capacity, S_E), Mna (malonate, CII inhibition), and Ama (antimycin A, CIII inhibition, Rox). Oxygen consumption was corrected for Rox. Blue plots indicate O₂ concentration, red plots are the O₂ consumption expressed per cell, and neon plots indicate safranin concentrations. (e) The Rox-corrected respiration and (f) the relative safranin signal: ROUTINE state, R, and in four respiratory states in permeabilized cells: S-linked LEAK respiration in the absence of adenylates, S_L(n), S-linked OXPHOS capacity, S_P, S-linked LEAK respiration after inhibition of ATP synthase by oligomycin, S_L(Omy), and S-linked ET capacity, S_E. Results are expressed as mean ± S.D. * denotes p < 0.05 while ** denotes p < 0.01 compared to NALCL as determined by Student’s t-test.

Figure 3

Succinate-linked respiration of NALCL and ALCL measured simultaneously with mitochondrial membrane potential in the presence of safranin. (a,b) are the protocols for measuring respiration in NALCL and ALCL, respectively, while (c,d) are the protocols for measuring mitochondrial membrane potential, mtMP using safranin concentration in NALCL and ALCL, respectively. Before the cells were added, safranin was calibrated up to 2 µM. The initial drop to 1.6 µM safranin is mainly due to unspecific binding upon the addition of cells. Low safranin concentrations indicate high mtMP. Titrations: Cell, dig (digitonin, cell permeabilization), Rot (rotenone, CI inhibition), S (succinate, S-LEAK respiration, S_L(n)), ADP (S-OXPHOS capacity, S_P), Omy (oligomycin, S-LEAK, S_L(Omy)), CCCP (S-ET capacity, S_E), Mna (malonate, CII inhibition), and Ama (antimycin A, CIII inhibition, Rox). Oxygen consumption was corrected for Rox. Blue plots indicate O₂ concentration, red plots are the O₂ consumption expressed per cell, and neon plots indicate safranin concentrations. (e) The Rox-corrected respiration and (f) the relative safranin signal: ROUTINE state, R, and in four respiratory states in permeabilized cells: S-linked LEAK respiration in the absence of adenylates, S_L(n), S-linked OXPHOS capacity, S_P, S-linked LEAK respiration after inhibition of ATP synthase by oligomycin, S_L(Omy), and S-linked ET capacity, S_E. Results are expressed as mean ± S.D. * denotes p < 0.05 while ** denotes p < 0.01 compared to NALCL as determined by Student’s t-test.